Abstract We propose to examine the characteristics and function of macrophages found in the pericardial cavity. We hypothesize that pericardial macrophages are a unique resident macrophage population that is able to traffic to the myocardium after cardiac injury and play a protective/reparative role, thus pericardial space is a reservoir of macrophages of significant regulatory physiologic importance. We present here in the preliminary data the first comprehensive immunophenotyping of pericardial macrophages in humans and mice. We obtained pericardial fluid of patients who underwent coronary artery bypass or valve replacement surgery. Murine pericardial fluid samples were obtained via a novel pericardial lavage technique pioneered in our lab. We show in our preliminary data that human pericardial macrophages are uniformly CD14+CD16+MHCII+CD206intCD116+CD11cneg CCR2neg while murine pericardial macrophages have a F4/80+CD64+MHCIIhighCD206intCD44+CD62+/-CCR2negMERTKnegLy6CnegCD11cneg CCR2negGATA6+ phenotype. The similar phenotypes indicate the high translational potential of murine studies in this project. Additionally, we have found that human and mouse pericardial macrophages secrete IL-10 and IL-6, suggesting a regulatory function. We have also found that pericardial macrophages traffic to the heart following myocardial infarction and can be distinguished from myocardial macrophages through their GATA6 expression. It is also clear from our preliminary data that pericardial macrophages differ from other serous cavity macrophages and therefore require independent study. In Aim 1 we will examine the fate of pericardial macrophages during myocardial infarction. We will induce myocardial infarction in GATA6Venus reporter mice and harvest pericardial fluid and pure myocardium at various time points to create a timeline for the trafficking of pericardial macrophages into the myocardium (Subaim 1.1). We will also perform parabiosis experiments by surgically joining CD45.1 GATA6Venus and CD45.2 LyzCreGATA6fl/fl. We will then induce myocardial infarction in the CD45.2 LyzCreGATA6fl/fl parabiont and harvest pericardial fluid and myocardium to examine whether GATA6+ macrophages are present in the CD45.2 LyzCreGATA6fl/fl parabiont, indicating replenishment by circulating monocytes (Subaim 1.2). In Aim 2 we will examine the protective function of pericardial macrophages following myocardial infarction. We will use both intra-pericardial injections of clodronate liposomes (Subaim 2.1) and LyzCreGATA6fl/fl mice (Subaim 2.2) to examine the effects of depleting pericardial macrophages during myocardial infarction. Cardiac function will be examined by echocardiography, EKG, size of scar after infarction by histology and the severity of inflammation via flow cytometry. Through our preliminary data we have discovered that the pericardial cavity is the niche for reparative cardiac macrophages. In this proposed project we will fully determine the origin and fate of these pericardial macrophages in murine models of ischemic heart disease. This project has high translational potential, as this population could be modulated with therapeutic purposes in humans.